Chagas disease is endemic throughout Mexico and Central and South
America, with [approximately equal to] 7.7 million persons infected,
108.6 million persons considered at risk, 3-3.3 million symptomatic
cases, an annual incidence of 42,500 cases (through vectorial
transmission), and 21,000 deaths every year (1-3). This disease is
caused by the protozoan parasite Trypanosoma cruzi, which is transmitted
to humans by blood-sucking insects of the family Reduviidae
(Triatominae). Although mainly a vector-borne disease, Chagas disease
also can be acquired by humans through blood transfusions and organ
transplantation (2-6), con genitally (from a pregnant woman to her baby)
(7), and through oral contamination, e.g., foodborne (8). Acute
infection can be lethal, and cardiomyopathy develops in 25%-30% of
infected persons (1). Although neither a vaccine against infection nor a
completely effective treatment for chronic Chagas disease currently
exists (2,9), treatment is now recommended for acute infections,
congenital infections, infections in immunosupressed persons, and
infections in children (10).

Although historically Chagas disease has been considered restricted
to Latin America (1,3), the disease is becoming a serious health issue
in the United States because of the presence of a notable number of
blood donors seropositive for T. cruzi (11-13). Notably, a small number
of the seropositive blood donors have never left the United States. Only
7 autochthonous cases of this disease have been reported in the United
States, all in the southern half of the country (14-19). The most recent
reported case of autochthonous transmission of T. cruzi occurred in 2006
near New Orleans, Louisiana (18). Many cases of Chagas disease in the
United States, however, may be overlooked because the early phase of the
infection is often asymptomatic (9,16), and health professionals are
largely unaware of this disease. In Arizona, humans may be at a greater
risk for vectorial transmission of the disease than previously thought
because human populations are rapidly expanding into habitats where
infected triatomines (20-22) and wild mammalian reservoirs such as
packrats, mice, armadillos, raccoons, and opossums (23-27) are
plentiful. Chagas disease is actively transmitted in domestic cycles
involving dogs in southern Texas (20,28), where >50% of triatomines
collected inside or near the homes of persons were found to be infected
with T. cruzi (19,20). Studies conducted many decades ago found that
triatomines in California, Arizona, and New Mexico were also infected
with T. cruzi (22-25,29).

Arizona is noteworthy as the state with the highest number of
triatomine-human contacts reported in the United States (American
Association of Poison Control Centers, www.aapcc.org/DNN; Arizona Poison
and Drug Information Center, University of Arizona Health Sciences
Center, www.pharmacy.arizona.edu/outreach/poison). In southern Arizona,
triatomine bugs live in association mostly with the white-throated
woodrat (Neotoma albigula) (24,26). Triatomine bugs have wingless
nymphal stages and winged adults. During their dispersal season
(beginning of May through July), adult insects, attracted by light,
reach human habitations (30-32). Triatoma rubida is by far the most
common species (Figure 1), but T. protracta and T. recurva are also
found (30,32). T. rubida was associated with a clinical case of Chagas
disease in the city of Guaymas, Mexico, although this bug is perhaps a
different subspecies than the one found in Arizona (33).

To our knowledge, the most recent comprehensive studies about the
infection rates by T. cruzi in triatomines from Arizona were conducted
>45 years ago (21,22), by using microscopy to detect the presence of
live parasites in the insect's gut or feces. In 1943, Wood (22)
found an overall infection rate of 4% in triatomines (28 of 699) from
Arizona collected over a 3-year period. In 1964, Bice (21) collected
triatomines from packrat dens in what is today a densely populated area
in metropolitan Tucson, Arizona, and found that 7.5% and 19.5% of T.
rubida and T. protracta bugs, respectively, were infected with T. cruzi
(21). A recent study that used molecular methods, but was based on a
small sample, found that 1 in 4 T. protracta and 0 of the 20 T. rubida
bugs examined were infected with T. cruzi (34).

To estimate the current potential of vectorial transmission of T.
cruzi disease in southern Arizona, we investigated the infection rate of
triatomines collected inside and around houses in metropolitan Tucson
(Pima County), Arizona. Tucson is the second largest metropolitan area
in Arizona with a population (as of 2007) of 1,003,235, of which 462,103
persons live in areas where triatomines are plentiful (35).

Materials and Methods

Collection of Insects

Triatomine insects were obtained by issuing public requests asking
residents of metropolitan Tucson (32[degrees]13'18"N,
110[degrees]55'35"W), Arizona, to collect bugs found inside or
around their houses. Insects that reach houses, as opposed to those
directly collected from nests of wild animals, are of greatest
epidemiologic importance because they have the highest chance of contact
with humans. Collectors were instructed to use a container and not to
touch or handle the insects with their bare hands, and they were usually
informed about the way that Chagas disease is transmitted. In a
preliminary study conducted in 2005, we found that some triatomine bugs
were infected with T. cruzi (C.E. Reisenman et al., unpub. data). We
therefore conducted a more extensive study in 2006. For each bug, we
recorded, whenever possible, the collection site (address), insect
species, stage, sex (if adults), and date of collection as well as any
other information the collector provided. Collected insects were
individually placed in 95% ethanol immediately after collection or upon
death and stored at 4[degrees]C until analysis. Insects were collected
during May 15-December 18, 2006.

[FIGURE 1 OMITTED]

Analysis of T. cruzi

Each insect was analyzed by PCR for the presence of T. cruzi.
Before analysis the insect was removed from ethanol and dried overnight
in a petri dish to remove traces of ethanol before DNA extraction. The
lower abdomen of each bug was detached with a sterile razor blade and
homogenized with a ceramic ball, or placed in a 1.5-mL microfuge tube
with phosphate-buffered saline ([less than or equal to] 80 [micro]L) and
homogenized with a hand-held mortar.

DNA was extracted following the instructions provided with the
QiaAmp DNA Blood Mini Kit (QIAGEN 51106; QIAGEN, Valencia, CA, USA). The
DNA was amplified by PCR according to an established T. cruzi
sample-processing protocol (36) by using the T. cruzi-specific primers
TCZ1 (5'-CGAGCTCTTGCCCACACGGGTGCT-3') and TCZ2
(5'-CCTCCAAGCAGCGGATAGTTCAGG-3'), which amplify 188 bp of a
repetitive nuclear sequence (15). For the minicircle locus, DNA was
amplified by using primers S35
(5'-AAATAATGTACGGGKGAGATGCATGA-3') and S36
(5'-GGGTTCGATTGGGGTTGGTGT-3') (37), which amplify a 330-bp
minicircle sequence. A 50-[micro]L reaction containing 0.4 [micro]M of
each primer, 20-40 ng of template DNA, and DNA polymerase (GoTaq;
Promega, Madison, WI, USA, or Platinum Taq; Invitrogen, Carlsbad, CA,
USA) was prepared. Primers for PCR were made at the Centers for Disease
Control and Prevention (Atlanta, GA, USA) core facility or acquired from
Invitrogen. The cycling parameters for the reactions with the TCZ1 and
TCZ2 primers were as described (36). The cycling parameters for the
reactions that used the S35 and S36 primers were an initial denaturation
at 95[degrees]C for 10 min, 35 cycles of amplification at 95[degrees]C
(30 s each), 58[degrees]C (30 s each) and 72[degrees]C (1 min each), and
a final extension at 72[degrees]C for 10 min. Samples were processed in
a Mastercycler Gradient Thermocycler Machine (Eppendorf, Hauppauge, NY,
USA) or an iCycler (Bio-Rad, Hercules, CA, USA). PCR products were
subjected to electrophoresis on 1.5% agarose gels, stained with ethidium
bromide, and visualized by using UV transillumination with AlphaImager
program (Alpha Innotech, San Leandro, CA, USA). All PCRs were run with a
positive control of known T. cruzi DNA and with a negative control in
which template DNA was omitted. Results that were positive for both sets
of primers were considered positive. If a sample was positive for only 1
set of primers, then the products of the PCR were cloned (pGem-T Easy
Vector System; Promega) and sequenced (Big Dye Terminator, v1.1 and ABI
31 30xl Genetic Analyzer; Applied Biosystems, Foster City, CA, USA).
Cloned sequences were compared with sequences in GenBank to determine if
the amplified sequence belonged to the T. cruzi genome. A random sample
of [approximately equal to] 15% negative samples (n = 11) was analyzed
along with positive samples to exclude the possibility of false-negative
samples.

Results

Insect Collection and Demographics

A total of 164 triatomine bugs (158 [96.3%] T. rubida,, 5 [3%] T.
recurva, and 1 [0.6%] T. protracta) were collected by volunteers and
analyzed for T. cruzi. Most of the collected T. rubida were adults
(93.6%, n = 151). Of the 141 adult T. rubida identified by sex, 87 were
females (62%) and 54 were males (38%). The proportion of females to
males was statistically different from a 1:1 sex ratio ([chi square] =
8.2, df = 1, p = 0.004).

Twenty-two collectors provided a total of 142 insects, with each
collector contributing a variable number of insects per night (range
1-10, median 2). A single collector provided 73 insects collected on 16
nights throughout the dispersal season. Twenty-two additional bugs were
collected by an unknown number of anonymous persons. Information about
the specific location where insects were collected was obtained for 84%
(n = 139 insects provided by 19 collectors) of the insects. These 139
insects (all T. rubida) were obtained from 17 collection sites
distributed in 6 of the 8 metropolitan Tucson areas corresponding to the
cardinal and ordinal points of the compass, and from 2 collection sites
in central Tucson (Table). Because insects were collected by volunteers
rather than by using systematic collection methods (i.e., light traps
set up in all geographic areas), the information in the Table serves the
sole purpose of reporting where insects were collected and does not
constitute an estimate of the abundance of insects per area.

Adult T. rubida insects were collected in or around houses from
mid-May through the end of August (Figure 2). Most adults were collected
in the last days of May and first week of June (Figure 2, panel B); a
total of 61% of insects were caught during May 25-June 8. This peak in
insect collections coincides with a typical, sustained increase in
minimum temperatures that enables insects to fly at night (32) (Figure
2, panel A). Bugs were collected steadily throughout the last week of
June; only 13 adults (8%) were collected during the rest of the
dispersal season, which extends to the end of August. Although insects
were not collected by using systematic methods, peak collection periods
coincide with the peak dispersals reported by Ekkens (32).

Analysis of Infection by T. cruzi

We found that 68 (41.5%) of the 164 bugs collected were infected
with T. cruzi. Twenty-four (35%) of the samples were positive by both
set of primers and therefore were considered positive. The remaining 44
(65%) positive samples were positive for S35/S36 only, but all of them
were confirmed positive by cloning and sequencing, thus excluding the
possibility of false-positive results. No samples were positive for
TCZ1/TCZ2 and negative for S35/S36.

Of the 22 identified sites or houses where insects were collected,
14 (63%) had at least 1 bug infected with T. cruzi. Infected bugs were
found in 7 of 8 areas, including central Tucson (Table). The percentage
of infected bugs per area was variable (median 43%, range 0%-100%),
likely due to the low number of bugs (1-2) collected in certain areas
(e.g., central, north, northeast). The mean [+ or -] SD percentage of
infected bugs per area, considering only those areas where >10
insects were collected, was 42.5% [+ or -] 1.0% (4 geographic areas, n =
132 insects). Similarly, to estimate the prevalence of infection per
collection site, we selected sites where at least 5 bugs were collected.
The mean [+ or -] SD number of infected bugs per collection site was
47.2% [+ or -] 5.7% (n = 7 collection sites in 4 geographic areas, n =
120 insects). This percentage was slightly higher (48.8 [+ or -] 6.6%, n
= 6 collection sites) when a site where a large number of bugs were
collected (n = 73) was excluded from the analysis.

The prevalence of infection by T. cruzi among triatomine species
was variable, as reported (21), although a larger sample is necessary to
confirm this prevalence. Forty-one percent of T. rubida (n = 158) bugs,
60% of T. recurva (n = 5) bugs, and the single T. protracta bug
collected were infected with T. cruzi. Because only a few T. recurva and
T. protracta bugs were collected, we restricted all further analysis to
T. rubida. Forty-two percent of nymphs (n = 7), 40.1% of females (n =
87), and 40.0% of males (n = 54) of T. rubida were found to be infected
with T. cruzi. Among adults, the probability of infection was
independent of sex ([CHI SQUARE] = 0.015, df = 1, p>0.9, by [chi
square] contingency analysis). Infected bugs were found throughout the
year; the median number of infected insects per 5-day collection period
during the dispersion season (mid-May through mid-July) was 27% (range
17%-67%).

Discussion

To our knowledge, almost no information has been collected during
the last half-century on the incidence of infection by T. cruzi in
triatomine bugs from Arizona (but see below). We found that 41.5% of the
164 collected bugs, most of which were T. rubida, were infected with T.
cruzi, and that 63% of houses or sites where insects were collected had
at least 1 specimen infected. Most bugs collected were adults, and this
winged life stage is known to be the main driver of dispersal (38).
Although most bugs were collected inside or around human houses from May
through the end of June, infected bugs were collected throughout the
period of study. Specimens of the less abundant species T. recurva and
T. protracta were also found to be infected. Samples that were positive
with only 1 set of primers were confirmed by sequencing of the amplified
DNA, excluding the possibility of false-positive results. In contrast
with our results and previous research by others (21,22), a recent study
found that none of the T. rubida bugs collected in the Tucson area were
infected with T. cruzi (34). This discrepancy might be explained by the
use of a different set of primers, the low numbers of insects examined
(n = 20 in the aforementioned study), or bias in the insect sample, such
as few collection sites. Furthermore, the infection rate reported here
is much higher than that reported in earlier studies in Arizona, which
ranged from 4% to 9% (22,24,29). Those studies were conducted by using
microscopy that visualized the presence of the parasite in the insect
gut; therefore, discrepancies maybe be attributed to differences in the
sensitivity of the methods used (e.g., 16).

[FIGURE 2 OMITTED]

The infection rates reported in this study, however, are in line
with those reported in other recent systematic studies. For instance,
51% of triatomines (mostly T. gerstaeckeri) collected from several areas
in Texas were infected (n = 241), with many insects found near human
dwellings (19). In Guaymas, in northwestern Mexico, 81% of T. rubida
collected in houses and in the peridomicile (n = 279) were infected with
T. cruzi (39). The fact that in that region adults and juveniles of T.
rubida were found inside houses indicates a progressive domiciliation of
this otherwise wild species, probably related to housing developments in
triatomine habitats (39). In our study, immature stage (nymphs) insects
collected inside houses were also infected, but the numbers are too
small to draw any definitive conclusions. If these houses are sites of
bug colonization, then the risk for human infection may be higher than
in houses where only adult insects were found and removed. Nevertheless,
because most immature insects in our study were found 1-4 months after
the peak of dispersion (i.e., they are likely the offspring of adults
that invade houses earlier) rather than consistently throughout the
year, T. rubida bugs do not appear to be in the process of becoming
domiciliated in Arizona.

Why have there been no reports of autochthonous cases of Chagas
disease in Arizona despite our finding that 41.5% of bugs are infected
with T. cruzi? In southern Arizona, triatomines live in close
association with the sylvatic animal reservoirs upon which they feed
(26) and apparently have a low capacity for domiciliation, although
juvenile insects (the offspring of dispersing adults) can be found in
houses near beds and readily feed on humans if necessary. Good housing
conditions (e.g., lack of crevices in walls or ceilings) do not favor
the permanent domiciliation of the insects, but this may not be the case
in rural areas where housing materials provide shelter for the insects.
Under those circumstances, colonization of human habitats might be
favored because at least half of dispersing adults were female and
likely gravid (C.E. Reisenman, unpub. data). In principle, the parasite
can be transmitted to humans when infected insects that invade houses
defecate on the skin of a human host upon feeding. Although a recent
study reported that T. rubida and T. protracta do not defecate while
feeding (34), our current investigations indicate that this is not the
case for T. rubida bugs in all stages and for both sexes (C.E.
Reisenman, unpub. data). Pet dogs can become infected by contamination
with excreta but also by contact with the oral mucosa when they
instinctively chew insects that might be infected (40).

Other reasons that might explain why Chagas disease is so rare in
the United States are the following: misdiagnosis of the early infection
(9,16), low insect vectorial capacity (34), or low infectivity of the
genetic lineage of the T. cruzi parasites present in local insects and
mammals, although this remains to be investigated. Bice (21) showed the
presence of T. cruzi parasites in the heart muscle of a mouse inoculated
with feces from an adult T. rubida bug collected in the Tucson area.
Should the lineage of T. cruzi present in southern Arizona correspond to
that associated with the pathogenic form of Chagas disease, the data
presented here suggest that vectorial transmission of the disease in the
area is possible.

DOI: 10.3201/eid1603.090648

Acknowledgments

We thank the 22 volunteer collectors for providing insects,
especially Phil Jenkins, Bill Savary, Jillian Savary, Robert Smith, and
Carl Olson. We also thank the Coordinating Center for Infectious
Diseases Core Facility at the Centers for Disease Control and Prevention
and C. Olson for identifying insects, Andrew Dacks for critically
reading this manuscript, and members of the Hildebrand laboratory for
helpful discussions.

This work was supported by an Arizona Biomedical Research
Commission grant no. 0708 (to J.G.H.).

(29.) Kofoid C, Whitaker B. Natural infection of American human
trypanosomiasis in two species of cone-nosed bugs, Triatoma protracta
Uhler and Triatoma Uhleri Neiva, in the western United States. J
Parasitol. 1936;22:259-63. DOI: 10.2307/3271533

Dr Reisenman is a researcher at the Department of Neuroscience at
the University of Arizona. Her research interests include vector biology
and sensory/neurophysiology of insect vectors of human and animal
diseases.